Animals ; DNA Damage ; DNA Repair ; DNA-Activated Protein Kinase ; Humans

BACKGROUND:It is accepted that the best method for spermatogonial stem cells separation is using artificial cryptorchism model combined with surface makers.OBJECTIVE:To explore the feasibility of separation spermatogonial stem cells with ?6-integrin and c-kit as specific surface markers.DESIGN,TIME AND SETTING:The randomized control experiment was performed at the Renmin Hospital of Wuhan University from May to December 2006.MATERIALS:Forty adult,white Kunming mice with 6 weeks old were randomly divided into cryptorchidism and control groups,with 20 animals in each group.METHODS:Artificial cryptorchidism model was prepared by made an incision at the median of abdomen,and testis was pulled into abdominal cavity,which was fixed at the each side of lateral abdominal wall.There was no treatment in the control group.The single cell suspension of seminiferous epithelium was obtained by traditional two step enzyme digestion at 2-3 months after operation.FITC-conjugated anti-?6-intergrin antibody and PE-conjugated anti-c-kit antibodies were added.Then the cells with low side scatter light-scattering properties were sorted and positively stained for ?6-intergrin and negative c-kit expression.Meanwhile,the viability of the isolated cells was assessed by trypan blue staining.MAIN OUTCOME MEASURES:The morphological changes of cryptorchidism,and the sorting results of spermatogonial stem cells.RESULTS:Cell distribution in seminiferous tubule was disorder with reduced numbers.The layer and lumens were disappeared,and cell division phase could be seen in the center of tubules.Compared to the control group,the testicular cells in the cryptorchidism group were increased in the side scatterlow,Forward scatterhi areas,with figure left-upward displacement.The distribution of ?6-integrin+ and c-kit cells were deviated each other,it named that most ?6-integrin+ cell were not spermatogonial stem cells,so do the c-kit-cells.Only 2.8% of testicular cells exhibited side scatterlow,?6-integrin+,and c-kit-,which were spermatogonial stem cells in the cryptorchidism group.And trypan blue staining showed that over 95% of them were viable.CONCLUSION:Using the two surface markers to sort spermatogonial stem cells can advance the purity of the spermatogonial stem cells in cell suspension,but the specificity is insufficient.

Objective Human Papillomavirus (HPV) is necessary to the development of cervical cancer.Until now,the distribution of specific HPV type has not been identified clearly yet.Besides,the relationship between age and the HPV type-specific distribution in cervical adenocarcinoma needs to be further investigated.Methods This study included 32 specimens of cervical adenocarcinoma out of a multicenter clinical study in China.The specimen blocks were sliced by sandwich method.DNA was amplified by SPF10-PCR method.DNA genotyping used LiPA method.All biopsy specimens had pathologic diagnosis.The prevalence of HPV type-specific distribution was described and the differences were analyzed statistically.Results 32 adenocarcinoma cases were included in this sttudy.HPV positive rate was 53.1％ (17/32).HPV-18 was the most prevalent type,which took up 47.4 ％ (8/17) of the total HPV positive cases,followed by HPV-16,which took up 26.3 ％ (5/17) of the total HPV positive cases.Other HPV types detected were HPV-39,HPV-45 and HPV-66.One multi-infection case was found (HPV-18 and HPV-66 co-infection).The average ages of HPV-16 and HPV-18 were 48.6 and 40.6 years old respectively,which showed statistically difference (P =0.049).When the patients younger than 50 years old were compared with the patients equal or older than 50 years old as two age groups,HPV positive rate did not show significantly statistical difference (P =0.074) between the two age groups.The prevalence of HPV-18 manifested significant differences between the two age groups in both HPV positive adenocarcinoma cases and overall adenocarcinoma cases (P =0.029and P =0.003 respectively).The prevalence of HPV-16 did not show any significant statistical difference between the two age groups in either HPV positive adenocarcinoma cases or overall adenocarcinoma cases (P =0.6 and P =1 respectively).HPV-16 and/or HPV-18 infection (at least one positive) in HPV positive adenocarcinoma cases did not have statistical significance (P =0.052),while in overall adenocarcinoma cases,HPV-16 and/or HPV-18 infection presented significant statistical difference (P =0.005).Conclusion HPV-18 and HPV-16 were two most prevalent HPV types in cervical adenocarcinoma.HPV infection and HPV-18infection were more common in young women.The prevalence of HPV-16 was more common in older adenocarcinoma cases compared with HPV-18.Those findings will provide evidence for the evaluation of HPV vaccine in preventing cervix related diseases.

Objective To study the inhibition of berberine on organ anion transporters (OATs) and its bidirectional trans-membrane transport.Method The transgene cell lines of the organ anion transporters including OAT1,OAT2,OAT3,OAT4,OAT7,and URAT1 were constructed and selected by animal cell transgenic method mediated by transporter Lipo 3000.Wild type (WT) cells were used as control group,and activity of OATs was verified by adding their radiolabeled substrates and inhibitors.The inhibition of 100 μmol/L berberine on the transporters was investigated in vitro.The IC50 of berberine on URAT1 was also determined.The bidirectional transport of berberine was studied through the Caco-2 model.Result The results showed that 100 μmol/L berberine inhibited the activity of OAT1,OAT2,OAT3,OAT4,OAT7 and URAT1 to (70.48±4.23)％,(69.13±1.28)％,(72.12±3.28)％,(79.77±6.49)％,(69.51 ±5.99)％ and (38.4 ± 2.67)％ respectively,the IC50 of berberine to URAT 1 was 13.19 μmol/L,the Papp (A-B) of 50 μmol/L and 100 μmol/L berberine were separately 0.28 × 10-6 and 0.40 × 10-6 cm/s,and the effiux rates were separately 3.18 and 3.15.Conclusion Berberine shows a stronger inhibition to URAT1 compared to OAT1,OAT2,OAT3,OAT4 and OAT7.Berberine may be the substrate of some effiux transporters.This study provides theoretical basis for explaining the low bioavailability ofberberine and forecasting the possible drug-drug interaction.

Objective To investigate the effects of Mycobacterium vaccae vaccine on immunological function and clinical character in elderly patients with stable chronic obstructive pulmonary disease (COPD). MethodsA total of 100 elderly patients with stable COPD were randomly divided into immunotherapy group (group A, n= 50) and non-immunotherapy group (group B, n= 50), and normal control group (group C, n = 50). The levels of peripheral blood T-lymphocyte subsets (CD3+ , CD4+, CD8+ , CD4+/CD8+ ratio), natural killer cells (NK cells), immunoglobulins (IgG, IgA, IgM) and cytokines (IL-6, IL-8, TNF-a) were measured respectively before and after therapy. Group A and B were followed up for 1 year, then the times of acute outbreak and hospitalization of patients in the two groups were also compared. Results The levels of CD4 + ,CD4+/CD8+ ratio and NK cells in group A, B were significantly lower before therapy (P＜0. 05～0. 01＝, and the levels of IL-6, IL -8, TNF-a and IgA were significantly higher than in group C (P＜0. 01＝. After treatment with Mycobacterium vaccae vaccine in group A, the levels of CD4+ , CD4+/CD8+ ratio and NK cells were significantly higher (P＜0. 05-0. 01＝ and IL-6, IL-8, TNF-a and IgA were significantly lower than before treatment (all P＜0. 01＝. These levels showed no significant changes in group B after treatment (P＞0. 05). After 1-year follow-up, the times of acute outbreak and hospitalization on patients were statistically lower in group A than in group B (P＜ 0. 01 ).ConclusionsMycobacterium vaccae vaccine can improve cellular immunity function and reduce the times of acute outbreak and hospitalization in patients with stable COPD, so it has a higher clinical application value.

OBJECTIVETo study the correlation between the expansion with different volume and the contents of the b-FGF and EGF in the expanded skin.

METHODSThe piglets were used in the experiment,which were divided into 7 groups including the sham, the blank control,the experimental groups with inflation of 1/2 volume, 3/4 volume, full volume, 150% volume and 200% volume. When expansion was complete, b-FGF was measured using enzyme coagulate method. EGF was measured by radioimmunoassay. The data were analyzed statistically.

RESULTSThe contents of b-FGF and EGF in the expanded skin was significantly higher than normal skin (P < 0.05). The difference in contents of b-FGF and EGF between the over-expanded skin and normal skin was more significant (P < 0.01).

CONCLUSIONExpansion can lead to the increase of b-FGF and EGF contents in the expanded skin.The biological effects may be relative to epidermal hyperplasia and angiogenesis of the expanded skin.

Animals ; Epidermal Growth Factor ; analysis ; metabolism ; Fibroblast Growth Factors ; analysis ; metabolism ; Models, Animal ; Radioimmunoassay ; Skin ; drug effects ; metabolism ; Swine ; Water ; administration & dosage

Objective To investigate the correlation between chronic insomniacs' sleep quality and age,gender,education level,anxiety,depression and sympathetic skin response (SSR) in chronic insomniacs.Methods General information of 197 outpatients with chronic insomnia was recorded,including age,gender and education,etc.They were tested by Pittsburgh's Sleep Quality Index (PSQI),Hamilton's Anxiety Scale (14 item version) (HAMA14),Hamilton's Depression Scale (24 item version)(HAMD24) and Sympathetic Skin Response (SSR).Distribution properties of different age,gender and education groups were studied.Chronic insomniacs were divided into mild insomnia group (7 ≤ PSQI ＜ 14)and moderate-severe insomnia group (PSQI ≥ 14).Dependency relation analysis and stepwise linear regression analysis were conducted among indices of PSQI scores,HAMA14 scores and total score,HAMD24 scores and total score,SSR positive incidence.Results Among 197 chronic insomniacs (male,50 cases,25.4％;female,147 cases,74.6％),insomniacs aged over 40 accounted for 77.2％.Female patients were older than male patients with statistical significance,of whom those aged 40-60 years accounted for the highest proportion of 37.1％.Female patients with less education (junior high school and below)accounted for the highest proportion of 50.3％ (73/197),whose education level was generally lower than male patients.Among 197 chronic insomniacs,104 cases (52.8％,99/197) had mild insomnia and 93cases (47.2％) had moderate-severe insomnia.Total score of HAMA14 of patients with moderate-severe insomnia was significantly higher than that of patients with mild insomnia (16.47 ± 5.40 vs 12.51 ± 4.53;t =5.552,P＜0.01).There was statistically significanct difference in subitem HAMA14 scores of anxiety somatization factor (4.31 ± 2.26 vs 5.90-3.10,t =5.600,P ＜ 0.01) and spiritualized anxiety factor (10.5 ± 72.97 vs 8.20 ± 3.00,t =4.157,P ＜ 0.01) between mild and moderate-severe groups with insomnia.Total score of HAMD24 of patients with moderate-severe insomnia was significantly higher than that of patients with mild insomnia (18.04 ± 5.91 vs 13.41 ± 5.05;t=3.931,P＜ 0.01).There was statistically significanct difference in scores of most HAMD24 subitems including anxiety/somatization (3.56 ± 1.51 vs 2.94 ± 1.28;t =3.110,P =0.002),cognitive dysfunction (2.91 ± 1.68 vs 2.17 ± 1.57;t=3.191,P=0.002),retardation (2.331 ±1.31 vs 1.72 ±1.22;t=3.939,P=0.01),dyssomnia (4.51 ± 1.54 vs 3.01 ± 1.80;t =6.228,P ＜0.01) and hopelessness factor (2.29 ± 1.46 vs 1.66 ± 1.07,t =3.459,P =0.001;except body weight and diurnal variation factor) between groups with different degrees of insomnia.SSR abnormal incidences of moderate-severe insomniacs were significantly higher than that of mild insomniacs.The proportion of poorly differentiated waveform and not elicited waveform in SSR abnormal groups had statistically significant difference.The Pearson correlation analysis showed that PSQI scores in chronic insomnia patients and HAMA14,HAMD24 score as well as abnormal rate of SSR were positively correlated (r =0.439,0.465,0.249,all P ＜ 0.01).Conclusions Chronic insomnia was commonly seen in middle-aged women with education level of junor high school and below.The degree of sleep quality and anxiety,depression as well as the abnormal rate of SSR was positively correlated in patients with chronic insomnia.

OBJECTIVETo investigate the relationship between the blood flow of the exceedingly expanded skin and the viability of the flap.

METHODSThe piglets were set up as the animal model. They were divided into 7 groups according to different injection volume of the tissue expander, i. e. injection to 1/2 content, 3/4 content, full content, 50% over content, 100% over content, sham-operation control and normal control. At different stages of expansion, the blood flow and the amplitude of the expanded skin were examined by laser Doppler. The survival length of the expanded flap was measured. Furthermore, statistical analysis was performed.

RESULTSAs the expansion volume increasing, the blood flow and the amplitude of the expanded skin increased, the survival length of the expanded flap increased also. In the meantime, the amplitude instability and vessel permeability increased. When the injection volume exceeded to 100%, the blood flow and the survival length of the expanded flap decreased.

CONCLUSIONSIn soft tissue and skin expansion, increasing injection volume could increase the survival length of the expanded flap, while over-expansion to 100% content may cause blood flow disturbance.

Animals ; Graft Survival ; Models, Animal ; Skin ; blood supply ; Skin Transplantation ; Surgical Flaps ; blood supply ; Swine ; Swine, Miniature ; Tissue Expansion Devices

Objective To study the inhibitory effects ofberberine on human organic cation transporter (OCTs) including OCT1,OCT2,OCT3,OCTN1 and OCTN2.Methods Using animal cell transgenic method mediated by transporter Lipo 3000,the drug transporters over expression cell lines S2-OCT1,S2-OCT2,S2-OCT3,S2-OCTN1 and S2-OCTN2 were obtained by selective medium culture.The OCTs evaluation model was established by detecting the trans-membrane transport of radioactive substrate in vitro.Wild type (WT) cells were used as control group,activity of OCTs was verified by adding its inhibitor.The inhibition of berberine on the transporters was investigated in vitro.The IC50 of inhibitory effect of berberine on various drug transporters was also calculated.Result The transport activity of transporter cell lines was increased by more than 5 times compared to the WT cell line respectively,what's more,their transport activity decreased significantly by their corresponding inhibitor.The ICs0 of berberine to OCT1,OCT2,OCT3,OCTN1 and OCTN2 were respectively 7.63,6.80,2.25,4.66 and 210.34 μmol/L.Conclusion Berberine significant inhibition to OCT1,OCT2,OCT3,OCTN1 and OCTN2.The inhibition on OCT1,OCT2,OCT3,OCTN1 is stronger compared to OCTN2.

Objective To evaluate 99Tcm-Arg-Glu-Ser (99Tcm-RES) as a potential tumor imaging agent. Methods RES was synthesized using solid phase peptide synthesis. The optimal labeling conditions of RES were determined under different reagents and reacting temperatures using SnC12 as reducing agent.The biodistribution of 99Tcm-RES was studied in nude mice bearing human lung cancer A549. Results The radiochemical purity of 99Tcm-RES was up to 85% and the radiochemical purity was 75% ever after 6 h at room temperature. The tumor uptake of 99Tcm-RES was obvious and the radioactivity ratios of tumor/blood,tumor/heart, tumor/liver, tumor/lung, tumor/spleen and tumor/muscle were 5.31, 1.88, 1.57, 3.58,4. 16 and 5.92, respectively at 6 h after 99Tcm-RES injection. Gamma camera imaging showed that tumor uptake of 99Tcm-RES was negative in rabbits with inflammatory mass but positive in those bearing tumor.The radioactivity ratio of tumor/inflammation was 3.12 at 6 h after injection. Conclusion 99Tcm-RES might possibly become a potential tumor imaging agent.